The human immunodeficiency virus (HIV-1) envelope glycoproteins (Envs) mediate the entry of the virus into the host cell. The Env spike is the only virus-specific component exposed on the virus surface and thus represents a key target for small-molecule entry inhibitors and vaccine-induced antibodies. The Env spike is a trimer consisting of three gp120 exterior Envs and three gp41 transmembrane Envs. To evade host neutralizing antibodies, the unliganded Env spike assumes a closed conformation (State 1). During the virus entry process, the high potential energy stored in the closed Env conformation is channeled into the eventual fusion of the viral and target cell membranes. The Env spike is opened by binding to the first receptor on the target cell, CD4, resulting in the formation of a pre-hairpin intermediate (State 3). Binding to the second receptor, CCR5 or CXCR4, leads to the formation of a very stable gp41 six-helix bundle (State 4) and the fusion of viral and host cel membranes. Recent evidence has suggested the existence of multiple relaxed intermediate states (States 2a, 2b, etc.) between the unliganded closed Env conformation (State 1) and the pre-hairpin intermediate (State 3). HIV-1 strains differ in their propensity to make the transition from State 1 to the relaxed intermediate states; more triggerable or reactive Envs are more likely to sample the relaxed intermediate states. These differences in Env reactivity influence HIV-1 tropism and sensitivity to inhibition by small molecules and antibodies. Thus, defining the conformational landscape available to HIV-1 Env variants is essential for understanding HIV-1 biology and for guiding the development of interventions. The overall goal of this proposal is to understand the conformational transitions of the HIV-1 Envs that are critical to virus entry. Each of the three Specific Aims investigates a different stage of the HIV-1 entry process: 1) Specific Aim 1 tests the hypothesis that modification of specific structures on the Env trimer releases the restraints that maintain conformational State 1, allowing Env to sample the relaxed intermediate states (States 2a, 2b, etc.). The effects of the Env changes on HIV-1 dependence on target cell receptor levels and sensitivity to inhibition by small molecules and antibodies will be determined. 2) Specific Aim 2 uses gp41 cysteine-substitution mutants that are dependent upon a reducing agent for infectivity to test the hypothesis that the gp41 coiled coil in the pre-hairpin intermedite (State 3) forms in an ordered fashion. Conformations of Env targeted by gp41-directed entry inhibitors will be identified. 3) Specific Aim 3 will define the Env changes and mechanism underlying HIV-1 adaptation to replicate in CD4-positive cells expressing very low levels of the CCR5 coreceptor. The above studies will illuminate the dynamic conformational transitions of the HIV-1 Env membrane-fusing machine and will expedite the development of inhibitors of HIV-1 entry.